Tobacco smoke-induced lung fibrosis and emphysema.

Despite public health campaigns discouraging smoking, 1,000 American children every day become smokers, ensuring that tobacco-related health complications will be with us for decades to come. Smoking is the greatest risk factor for both chronic obstructive lung disease and interstitial lung disease. The facts that not every smoker develops chronic lung disease and that lung pathology differs markedly among smokers indicate that individual susceptibility must be a central determinant of lung injury responses to cigarette smoke. Comparative examination of pathogenic mechanisms of smoke-induced lung disease can shed light on the homeostatic pathways critical to maintaining lung health. In this review, we explore common and divergent biological forces tilting the lung homeostatic balance away from health and toward emphysema or pulmonary fibrosis. We emphasize recent insights that highlight the greatest contrasts or similarities in the pathogenesis of these two chronic lung disease phenotypes.

Transforming growth factor-ß1 downregulates vascular endothelial growth factor-D expression in human lung fibroblasts via the Jun NH2-terminal kinase signaling pathway.

Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-ß1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-ß1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-ß1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-ß1 effect can be abolished by inhibitors of TGF-ß type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-ß1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.

Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-ß receptor I internalization and inhibiting transforming growth factor-ß1 signaling.

RATIONALE: Alveolar transforming growth factor (TGF)-ß1 signaling and expression of TGF-ß1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-ß receptor TßRI inhibits TGF-ß signaling and could attenuate development of experimental lung fibrosis. OBJECTIVES: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-ß1 signaling in alveolar epithelial cells. METHODS: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-ß1 signaling, TGF-ß1, and TßRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. MEASUREMENTS AND MAIN RESULTS: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-ß1 signaling and downstream expression of TGF-ß1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-ß1 and TßRI in alveolar epithelial cells, which inhibited TGF-ß1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. CONCLUSIONS: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-ß1 and TßRI internalization and inhibiting TGF-ß1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TßRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

Regulation and functional significance of autophagy in respiratory cell biology and disease.

Autophagy is a homeostatic process common to all eukaryotic cells that serves to degrade intracellular components. Among three classes of autophagy, macroautophagy is best understood, and is the subject of this Review. The function of autophagy is multifaceted, and includes removal of long-lived proteins and damaged or unneeded organelles, recycling of intracellular components for nutrients, and defense against pathogens. This process has been extensively studied in yeast, and understanding of its functional significance in human disease is also increasing. This Review explores the basic machinery and regulation of autophagy in mammalian systems, methods employed to measure autophagic activity, and then focuses on recent discoveries about the functional significance of autophagy in respiratory diseases, including chronic obstructive pulmonary disease, cystic fibrosis, tuberculosis, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, acute lung injury, and lymphangioleiomyomatosis.

Retinoic acid-related orphan receptor-α is induced in the setting of DNA damage and promotes pulmonary emphysema.

RATIONALE: The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. OBJECTIVES: To determine the role of Rora in smoke-induced emphysema. METHODS: Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. MEASUREMENTS AND MAIN RESULTS: Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. CONCLUSIONS: Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

Statins and pulmonary fibrosis: the potential role of NLRP3 inflammasome activation.

RATIONALE: The role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) in the development or progression of interstitial lung disease (ILD) is controversial. OBJECTIVES: To evaluate the association between statin use and ILD. METHODS: We used regression analyses to evaluate the association between statin use and interstitial lung abnormalities (ILA) in a large cohort of smokers from COPDGene. Next, we evaluated the effect of statin pretreatment on bleomycin-induced fibrosis in mice and explored the mechanism behind these observations in vitro. MEASUREMENTS AND MAIN RESULTS: In COPDGene, 38% of subjects with ILA were taking statins compared with 27% of subjects without ILA. Statin use was positively associated in ILA (odds ratio, 1.60; 95% confidence interval, 1.03-2.50; P = 0.04) after adjustment for covariates including a history of high cholesterol or coronary artery disease. This association was modified by the hydrophilicity of statin and the age of the subject. Next, we demonstrate that statin administration aggravates lung injury and fibrosis in bleomycin-treated mice. Statin pretreatment enhances caspase-1-mediated immune responses in vivo and in vitro; the latter responses were abolished in bone marrow-derived macrophages isolated from Nlrp3(-/-) and Casp1(-/-) mice. Finally, we provide further insights by demonstrating that statins enhance NLRP3-inflammasome activation by increasing mitochondrial reactive oxygen species generation in macrophages. CONCLUSIONS: Statin use is associated with ILA among smokers in the COPDGene study and enhances bleomycin-induced lung inflammation and fibrosis in the mouse through a mechanism involving enhanced NLRP3-inflammasome activation. Our findings suggest that statins may influence the susceptibility to, or progression of, ILD. Clinical trial registered with www.clinicaltrials.gov (NCT 00608764).

Codon-optimized Luciola italica luciferase variants for mammalian gene expression in culture and in vivo.

Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-d-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.

Carbon monoxide modulates alpha-smooth muscle actin and small proline rich-1a expression in fibrosis.

Carbon monoxide (CO) is a biologically active molecule produced in the body by the stress-inducible enzyme, heme oxygenase. We have previously shown that CO suppresses fibrosis in a murine bleomycin model. To investigate the mechanisms by which CO opposes fibrogenesis, we performed gene expression profiling of fibroblasts treated with transforming growth factor-beta(1) and CO. The most highly differentially expressed categories of genes included those related to muscular system development and the small proline-rich family of proteins. We confirmed in vitro, and in an in vivo bleomycin model of lung fibrosis, that CO suppresses alpha-smooth muscle actin expression and enhances small proline-rich protein-1a expression. We further show that these effects of CO depend upon signaling via the extracellular signal-regulated kinase pathway. Our results demonstrate novel transcriptional targets for CO and further elucidate the mechanism by which CO suppresses fibrosis.

Cross talk between Id1 and its interactive protein Dril1 mediate fibroblast responses to transforming growth factor-beta in pulmonary fibrosis.

The presence of activated fibroblasts or myofibroblasts represents a hallmark of progressive lung fibrosis. Because the transcriptional response of fibroblasts to transforming growth factor-beta(1) (TGF-beta(1)) is a determinant of disease progression, we investigated the role of the transcriptional regulator inhibitor of differentiation-1 (Id1) in the setting of lung fibrosis. Mice lacking the gene for Id1 had increased susceptibility to bleomycin-induced lung fibrosis, and fibroblasts lacking Id1 exhibited enhanced responses to TGF-beta(1). Because the effect of Id1 on fibrosis could not be explained by known mechanisms, we performed protein interaction screening and identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and was recently implicated in TGF-beta(1) signaling during embryogenesis. To date, little is known about the function of Dril1 in humans. Although it has not been previously implicated in fibrotic disease, we found that Dril1 was highly expressed in lungs from patients with idiopathic pulmonary fibrosis and was regulated by TGF-beta(1) in human fibroblasts. Dril1 enhanced activation of TGF-beta(1) target genes, whereas Id1 decreased expression of these same molecules. Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1.

Protective functions of heme oxygenase-1 and carbon monoxide in the respiratory system.

The respiratory system, including the lung and upper airways, succumbs to injury and disease through acute or chronic exposures to adverse environmental agents, in particular, those that promote increased oxidative or inflammatory processes. Cigarette smoke and other forms of particulate or gaseous air pollution, allergens, microorganisms infections, and changes in inspired oxygen may contribute to lung injury. Among the intrinsic defenses of the lung, the stress protein heme oxygenase-1 constitutes an inducible defense mechanism that can protect the lung and its constituent cells against such insults. Heme oxygenases degrade heme to biliverdin-IXalpha, carbon monoxide, and iron, each with candidate roles in cytoprotection. At low concentrations, carbon monoxide can confer similar cyto and tissue-protective effects as endogenous heme oxygenase-1 expression, involving antioxidative, antiinflammatory, antiproliferative, and antiapoptotic effects. Lung protection by heme oxygenase-1 or its enzymatic reaction products has been demonstrated in vitro and in vivo in a number of pulmonary disease models, including acute lung injury, cigarette smoke-induced lung injury/chronic obstructive pulmonary disease, interstitial lung diseases, ischemia/reperfusion injury, and asthma/airway inflammation. This review summarizes recent findings on the functions of heme oxygenase-1 in the respiratory system, with an emphasis on possible roles in disease progression and therapies.

Carbon monoxide: to boldly go where NO has gone before.

The discovery that nitric oxide (NO) has powerful vasoactive properties identical to those of endothelial-derived relaxing factor spawned a vast body of research investigating the physiological actions of small gas molecules. NO, which arises endogenously through the action of nitric oxide synthase (NOS) enzymes, is a highly reactive gas that plays important roles in the regulation of vascular and immune function. Carbon monoxide (CO), a similar yet much more chemically stable gas, occurs in nature as a product of the oxidation or combustion of organic materials. CO also arises in cells and tissues as a byproduct of heme oxygenase (HO) activity, which degrades heme to biliverdin-IXalpha. Like NO, CO acts as a vasorelaxant and may regulate other vascular functions such as platelet aggregation and smooth muscle proliferation. CO has also been implicated as a neurotransmitter in the central nervous system. HO-1, the inducible form of HO, confers cytoprotection against oxidative stress in vitro and in vivo. CO, when applied at low concentration, exerts potent cytoprotective effects mimicking those of HO-1 induction, including down-regulation of inflammation and suppression of apoptosis. Many of the effects of CO depend on the activation of guanylate cyclase, which generates guanosine 3',5'-monophosphate (cGMP), and the modulation of mitogen-activated protein kinase (MAPK) signaling pathways. This review highlights new advances in the interaction of CO with cellular signaling processes.

Epithelial cell mitochondrial dysfunction and PINK1 are induced by transforming growth factor-beta1 in pulmonary fibrosis.

BACKGROUND: Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis. METHODS: We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-ß1 (TGF-ß1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis. RESULTS: Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-ß1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis. CONCLUSION: TGF-ß1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.

Carbon monoxide and human disease.

Carbon monoxide is produced endogenously in humans through the breakdown of hemoglobin by heme oxygenase. Although originally thought to be a superfluous by-product of heme catabolism, carbon monoxide is now known to play a central role in many aspects of human health and disease. The functions of carbon monoxide that have been described to date are myriad, including blood pressure regulation, maintenance of organ-specific vascular tone, neurotransmission, stress response, platelet activation, and smooth muscle relaxation. This review outlines what is known to date about carbon monoxide as it relates to human disease.

Carbon monoxide-dependent signaling.

It has become accepted that nitric oxide serves important functions in biological systems as a second messenger. Another diatomic gaseous molecule, carbon monoxide (CO), is also rapidly gaining acceptance as a signaling agent. Some of the activities of CO are analogous to those of nitric oxide in the vascular system and the brain, but CO also behaves in novel ways. Like nitric oxide, CO is capable of activating soluble guanylyl cyclase. This mechanism of CO signaling is important in vasodilation and neurotransmission. There is growing evidence, however, that CO also acts independently of soluble guanylyl cyclase. CO has been shown to protect against septic shock and lung injury in animal models, and the mitogen-activated protein kinase system appears to mediate this cytoprotective effect. Although much remains to be elucidated about the mechanisms of cell signaling by CO, the pace of discovery in this field is making the picture clearer with every passing day.

The expanding role of biomarkers in the assessment of smoking-related parenchymal lung diseases.

Recent advances in the field of clinical biomarkers suggest that quantification of serum proteins could play an important role in the diagnosis, classification, prognosis, and treatment response of smoking-related parenchymal lung diseases. COPD and idiopathic pulmonary fibrosis (IPF), two common chronic progressive parenchymal lung diseases, share cigarette smoke exposure as a common dominant risk factor for their development. We have recently shown that COPD and interstitial lung disease may represent distinct outcomes of chronic tobacco use, whereas others have demonstrated that both diseases coexist in some individuals. In this perspective, we examine the potential role of peripheral blood biomarkers in predicting which individuals will develop COPD or IPF, as well as their usefulness in tracking disease progression and exacerbations. Additionally, given the current lack of sensitive and effective metrics to determine an individual's response to treatment, we evaluate the potential role of biomarkers as surrogate markers of clinical outcomes. Finally, we examine the possibility that changes in levels of select protein biomarkers can provide mechanistic insight into the common origins and unique individual susceptibilities that lead to the development of smoking-related parenchymal lung diseases. This discussion is framed by a consideration of the properties of ideal biomarkers for different clinical and research purposes and the best uses for those biomarkers that have already been proposed and investigated.

Autophagy in idiopathic pulmonary fibrosis.

BACKGROUND: Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. METHODS: Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-ß(1) on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. RESULTS: Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-ß(1) inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-ß(1). In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. CONCLUSION: Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-ß(1) may represent a mechanism for the promotion of fibrogenesis in IPF.